DSBs IN REAL TIME[unreadable] Our approaches to addressing DSB induction and repair have been extended to consider repair in the context of chromosomes. While DNA is the central component of chromosomes, the relationship between DNA and chromosome breaks (CRBs) has not been addressed nor the dynamics of DSB repair in different chromosomes. In spite of many genetic and biochemical assays for checkpoint arrest and repair, little is known about the behavior of damaged chromosomes in damage-arrested cells. Furthermore, the question of the relationship between a DSB in DNA and a cytologically detectable CRB, as well as possible genetic controls have never been addressed. We developed a system based in the yeast Saccharomyces cerevisiae that provides for chromosome analysis in real time following the induction of a single site-specific DSB by an I-SceI endonuclease under the tight control of a GAL1promoter. We utilized tetR-CFP and LacI-GFP sequence binding proteins to mark each side of a DSB and Spc29-RFP fusion to identify the spindle poles. These proteins bind multiple repeats of their operater target sequences. This allows us to investigate the development of a chromosome break following DNA scission and the relation to spindle pole separation and sister chromatid separation in wild type and various Ku and Rad50/MRE11/XRS2 (R/M/X) mutants. We have established that the transition from DNA double-strand break measured at the molecular level to a cytologically detectable chromosome break is prevented by the physical tethering function of the R/M/X complex and that the appearance of a chromosome break in vivo requires force that is transmitted through microtubules. Specifically, we found that induction of a single DSB led to the accumulation of cells in G2/M with extended spindle poles, but no sister chromatid separation. Although there was induction of a molecularly detectable DSB, there was no cytologically detectable chromosome break (CRB) in wild type cells, based on the lack of separation of the markers at each side of the DSB. Instead, a CRB is only revealed in mutants. The R/M/X complex prevents the manifestation of a CRB from a DSB. Moreover, the prevention of CRBs depends on the structural rather than the nuclease features of Mre11 complex. The production of a CRB requires force that is transmitted through microtubules. While a rad50 mutant exhibits chromosome breaks, the level is only 10 to 15% of cells, even though nearly ever cell has a DSB. This has suggested another component is holding broken chromosomes together. Surprisingly, there is a cold sensitive component, which in rad50 mutants at low temperatures results in nearly 40% of cells having a chromosome break. We are currently pursuing this additional component as well as assessing the role of centromere forces and also sister chromatid cohesion. We have now established that cohesin can play a role in holding the broken ends of chromosomes together. We reasoned that end processing might be important, leading us to investigate a possible role of exonuclease. Importantly, we have have established that there is an exonuclease that is essential in preventing the transition from DSB to chromosome break, supporting our view that resected DNA may provide the DNA scaffold to prevent transition from a DNA to a chromosome break. [unreadable] [unreadable] GENERAL REPAIR OF DSBS IN THE CELL[unreadable] Since chromosomes can be displayed as individual bands according to size using pulse-field gel electrophoresis(PFGE), we reasoned that it would be possible to address repair in individual chromosomes and genetic controls. We assessed the randomness of DSB production as well as repair. Over the range of sizes from 250 to 1500 MB, induction of DSBs by ionizing radiation is random with a yield of approximately 0.6 breaks/100 Gy/Mb (or 1 DSB/haploid G1 cell/10 Gy. By comparing the restitution of full length chromosomes and the patterns of broken molecules following exposure to various doses we are now in a position to evaluate the extent to which DSB repair is random. There was little if any DSB repair leading to the restitution of full size chromosomal molecules in G1 diploid cells, except for Chromosome XII, while G2 cells exhibited efficient repair. Although a homologue was present that should allow for interchromosomal recombinational repair in G1 cells, there appears to be restrictions on this repair during this phase of the cell cycle. The Chromosome XII difference in repair is likely related to much of it being composed of ribosomal DNA repeats. We propose that DSBs in these repeats can be repaired by a Rad51 independent, single-strand annealing mechanism rather than exchange mechanisms. These results suggest that many of the components necessary for DSB repair are present including the capability for resection. We found that DSBs may be repaired in a random fashion. Repair of DSBs induced in G2 cells by ionizing radiation was rapid, over 90% of chromosomes I being restituted in about 1.5 hours. The repair requires the RAD50, RAD51 and RAD52 genes, the larger chromosomes taking longer to be repaired, presumably because of the greater number of breaks. Surprisingly, we found that in various repair deficient mutants, that the chromosomes appeared to get larger based on PFGE analysis. In fact, we found that that the apparent increase in mass was due to resection (i.e., a decrease in mass) at the ends that resulted in slower movement in the PFGE. We have now explored this and established kinetics of resection and are assessing the roles of various genes on this process. Our findings suggest that resection at randomly induced breaks produced by ionizing radiation may be much different than for site-specific DSBs.[unreadable] [unreadable] DSBS AND GENOME REARRANGEMENTS. [unreadable] We have examined individual colonies arising from cells subjected to ionizing radiation (to induce DSBs)for the appearance of chromosome aberrations using CGH (Comparative Genome Hybridization) and found that nearly 30-50% of survivors irradiated in G-2 have an aberration. Through detailed analysis of individual chromosome events we have established that nearly all the breakpoints involve small repeats, either 5kb or 300 bp. Thus, while repair between sister chromatids is highly efficient, these findings demonstrate that repeats in the genome can compete effectively for a DSB containing region and have led us to conclude that Double strand breaks associated with repetitive DNA can unlock genome plasticity, the title of our recently submitted paper to one of the top journals. The distribution of aberrations and aneuploidy does not indicate differences in repair strictly on the basis of chromosome size.